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Notch-1 regulates Akt signaling pathway and the expression of cell cycle regulatory proteins cyclin D1, CDK2 and p21 in T-ALL cell lines
Leukemia Research, 5, 33, pages 678 - 685
Gain-of-function mutations in Notch-1 are common in T-cell lymphoblastic leukemia (T-ALL), making this receptor a promising target for drugs such as gamma-secretase inhibitors (GSIs). However, GSIs seem to be active in only a small fraction of T-ALL cell lines with constitutive Notch-1 activity and the downstream response of Notch signaling is only partially understood. To further investigate the molecular mechanisms underlying proliferation suppression and apoptosis and explore effective downstream target genes, we used RNA interference (RNAi) technology to down-regulate the expression of Notch-1 in GSIs-resistant T-ALL cell lines. Results showed that down-regulation of Notch-1 by transfection of a small interfering RNA (siRNA) could cause SupT1 cells proliferation inhibition by inducing G0/G1 cell cycle arrest and apoptosis. The proliferation inhibitory and apoptotic effects resulting from down-regulation of Notch-1 may be mediated through regulating the expression of cell cycle regulatory proteins cyclin D1, CDK2 and p21 and the activity of Akt signaling. In addition, our results demonstrated that down-regulation of Notch-1 signaling could sensitize SupT1 cells to adriamycin. Taken together, cell cycle regulatory proteins and Akt signaling may be attractive targets in T-ALL.
Keywords: Notch-1, T-cell acute lymphoblastic leukemia, Apoptosis, siRNA, Akt.
T-ALL is a malignant disease, constituting a substantial fraction of ALL, both in children and in adults. Current treatment is primarily based on combination chemotherapy with low long-term survival rate particularly in adult patients, emphasizing the need for improved therapy. The molecular mechanisms underpinning T-ALL are likely to be complex  and .
Deregulation of the Notch-1 signaling pathway has recently emerged as an important genetic component in T-ALL  and . The involvement of Notch-1 was first observed in a rare t(7;9) (q34;q34.3) translocation, which brings an activated form of the Notch-1 receptor gene under the control element of the T-cell receptor gene  . More recently, it was shown that more than 50% of all T-ALL patients carried Notch-1 gain-of-function mutations that generate an activated form of Notch  . Mutant Notch-1 could represent an important new target for therapy of T-ALL patients. Since the generation of activated Notch-1 can be inhibited by gamma-secretase inhibitors (GSIs)  , GSIs are valuable tools for delineating the cell biological function of the Notch cascade, but the efficacy of this strategy has been questioned, as GSIs seem to be active in only a small fraction of human and mouse T-ALL cell lines with constitutive Notch-1 activity , , , and . Therefore, it is important to explore more effective downstream target genes.
Although there is a wealth of data supporting the notion that the intracellular domain (ICD) of Notch in specific cellular contexts affects many downstream genes via CBF1/Suppressor of Hairless/Lag1 (CSL) to exert biological effect  and , the downstream response of Notch signaling is only partially understood. In T-ALL, our understanding of which downstream genes are activated, and how these affect cell cycle regulation and lead to cellular transformation, is rather limited. In keeping with this, there is evidence showing that c-MYC is an important new direct target gene  and  but not activated in all T-ALL cell lines  , suggesting the existence of other immediate Notch downstream effectors.
To investigate the role of Notch signaling, it is important to choose an appropriate tool. In practice, GSIs represent the most immediately promising therapeutic approach. However, several problems need to be considered in designing successful GSIs treatment for T-ALL. First, GSIs are not specific for Notch-1 and turn off all four Notch receptors  and . Furthermore, gamma-secretase might affect other proteins involved in proliferation  . In the present study, we used siRNA to down-regulate Notch-1 gene expression in the GSIs-resistant T-ALL cell lines SupT1 and Jurkat , , and . Our results demonstrated that the down-regulation of Notch-1 gene expression by siRNA could inhibit cell proliferation, induce G0/G1 cell cycle arrest and apoptosis, and this “Notch off” state was associated with reduced CDK2 and cyclin D1, increased p21 expression and disrupted Akt signaling. In addition, we demonstrated down-regulation of Notch signaling could increase T-ALL cell sensitivity to adriamycin. The results imply that cell cycle regulatory proteins and Akt signaling may be attractive targets in T-ALL.
2. Materials and methods
2.1. Cell culture
Human T-ALL cell lines SupT1 and Jurkat were grown in suspension in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 15% fetal-calf serum (FCS) (Gibco BRL, Grand Island, NY, USA), penicillin (100U/ml), streptomycin (100 μg/ml), and 2 mM l-glutamine (Gibco BRL, Grand Island, NY, USA). Cells were maintained in a humidified incubator at 37 °C and 5% CO2.
2.2. siRNA transfection
For Notch-1, sense siRNA is 5′-ACGAAGAACAGAAGCACAAAGGCGG-3′ and antisense siRNA is 5′-CCGCCUUUGUGCUUCUGUUCUUCGU-3′. For scrambled control, sense and antisense siRNAs are 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′, respectively. Notch-1 siRNA (Invitrogen) or the scrambled control siRNA (Invitrogen) was transfected at the final concentration of 100 nM oligonucleotide using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's recommendations. To enhance knock-down efficiency, we conducted the second transfection 24 h after the first. The cells were used for the following experiments.
2.3. Analysis of proliferation by MTT assay
Cells were planted at a density of 5000 cells/well in 96-well plates, and subsequently transfected with Notch-1 siRNA or control siRNA. 24, 48, 72 and 96 h after tansfection, 20 μl of MTT (Amresco, Cleveland, OH) was added per well. After an additional 4 h incubation, the media was removed, 200 μl of DMSO was added per well, the plate was mixed thoroughly for 10 min, and color development was measured on a microplate reader at 570 nm. For the chemotherapeutic sensitivity assay, 24 h after transfection, cells were exposed to different concentration of adriamycin (Sigma) for an additional 48 h and the MTT assay was performed as described above. The IC50 was calculated using the following formulas: Y1 − Y2/X1 − X2 = M; Y1 − MX1 = B; B − 50/−M = IC50, where (X1, Y1) and (X2, Y2) are two points below and above 50% inhibition rate (X = drug concentration and Y = % inhibition rate)  .
2.4. Real-time reverse transcription-PCR
Total RNA was isolated by Trizol (Invitrogen) according to the manufacturer's instructions. Approximately, one μg of total RNA from each sample was subjected to first-strand cDNA synthesis using RevertAid™ First Strand cDNA Synthesis Kit (MBI, Fermentas, USA). Reverse transcription reaction was done at 42 °C for 1 h, followed by 95 °C for 5 min. Real-time PCR was conducted using the Light-Cycler rapid thermal cycler system 2.0 (Roche Diagnostics Ltd., UK) in accordance to the manufacturer's instructions. The real-time PCR contained, in a final volume of 20 μl, 10 μl of 2× SYBR Green Real-time PCR Master Mix, 1 μl of cDNA, and 1 μl of the forward and reverse primers. The thermal cycling conditions were initial denaturation at 95 °C for 10 s, followed by 45 cycles of denaturation at 95 °C for 0 s, annealing at 59 °C for 5 s, and elongation at 72 °C for 10 s. The relative concentrations of the PCR products derived from the target gene were calculated using LightCycler System software. The primers used for the amplification of human Notch-1 and β-actin were shown in Table 1 .
|Target gene||Primer sequence|
2.5. Cell cycle analysis
The cell cycle distribution was determined by flow cytometric analysis of propidium iodide (PI) (BD ParMingen, San Diego, CA)-stained nuclei. After siRNA transfection, cells were collected, washed twice with PBS and fixed with 500 μl 75% cold ethyl alcohol in the dark overnight at 4 °C. Then cells were washed with PBS and incubated with 500 μl PI in the dark for 15 min at room temperature. Following staining, cells were immediately analyzed by flow cytometry. For the chemotherapeutic drug assays, 24 h after transfection, cells were exposed to adriamycin (final concentration 0.1 μg/ml) for an additional 48 h, and then cells were collected and analyzed as above. Cells with sub-G1 DNA content were considered apoptotic.
2.6. Apoptosis assay
Apoptosis induction was confirmed using the Annexin V/PI Apoptosis Detection Kit (Jingmei Biotech, Shenzhen, China). After siRNA transfection, cells were collected and washed twice with cold PBS. Cells were labeled with 5 μl Annexin V followed by 10 μl PI. Annexin V-PI were measured by FACS Calibur and analyzed with the Modfit Software.
2.7. Western blot analysis
Cells were lysed in lysis buffer [50 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.5% Triton X-100, 2.5 mM sodium orthovanadate, 10 μl/ml protease inhibitor cocktail, 1 mM phenylmethylsulfonyl fluoride] for 20 min at 4 °C. The protein concentrations were determined with the BCA Protein Assay reagent (Pierce, Rockford, IL), according to the manufacturer's instructions. Total proteins were fractionated by SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were blocked with blocking buffer [0.1 M Tris (pH 7.5), 0.9% NaCl, and 0.05% Tween-20 (TBST) containing 5% nonfat milk powder], then incubated with appropriate primary antibodies against cyclin D1 (CST, UK), CDK2 (CST, UK), and p21 (CST, UK), Notch-1 (Abcam Ltd., Cambridge, UK), HES-1 (Abcam Ltd., Cambridge, UK), phospho-Akt (Ser473) (CST, UK), Akt (CST, UK), followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated antibodies. The probed proteins were detected using the chemiluminescent reagents (SuperSignal West Pico West Chemiluminescent Substrate, Pierce, Rockford, IL). The bidimensional absorbance of proteins on the films were quantified and analyzed with Molecular Analyst software (Bio-Rad).
2.8. Statistical analysis
The data were reported as mean ± S.D. Differences among three groups were determined by analysis of one-way ANOVA variance, followed by Newman–Keuls test for multiple comparisons, whereas differences between two groups were evaluated by the Student's t-test for analysis of completely randomized two-group designs. P values less than 0.05 were considered statistically significant. Statistical analysis was performed by the SPSS 13.0 (SPSS Inc., Chicago, USA) statistical software programs.
3.1. Notch-1 siRNA effectively down-regulates Notch-1 expression
We initially examined the expression level of Notch-1 in T-ALL cell lines SupT1 and Jurkat by real-time RT-PCR and Western blot. The levels of Notch-1 mRNA expression showed a remarkable decrease in Notch-1 siRNA-transfected group in SupT1 and Jurkat cell lines ( Fig. 1 ). Western blot analysis showed that protein level of Notch-1 was greatly reduced in Notch-1 siRNA-transfected group compared with control siRNA-transfected group in both cells lines ( Fig. 4 A and B). To verify that the reduction of Notch-1 by siRNA was sufficient to block Notch-1 pathway, HES1 expression was analyzed. Down-regulation of Notch-1 expression by siRNA led to a decrease in HES1 protein expression in SupT1 and Jurkat cells ( Fig. 4 A and B).
3.2. Down-regulation of Notch-1 expression inhibits cell proliferation in SupT1 cells
Next we analyzed the effect of Notch-1 siRNA on cell proliferation. 72 h after transfection with Notch-1 siRNA or control siRNA, cell viability was detected by MTT assay. Down-regulation of Notch-1 expression by siRNA markedly inhibited cell proliferation in SupT1 cells ( Fig. 2 A), whereas no apparent changes in Jurkat cells occurred ( Fig. 2 B).
3.3. Down-regulation of Notch-1 expression induces G0/G1 phase cell cycle arrest in SupT1 cells
As cell proliferation is closely related to cell cycle progression, we analyzed the effects of Notch-1 siRNA on cell cycle distribution. Down-regulation of Notch-1 expression by siRNA led to a marked arrest in cell cycle progression, as characterized by an accumulation of cells in G0/G1 in SupT1 cells ( Fig. 3 A and C). No marked difference in cell cycle distribution was observed between Notch-1 siRNA-transfected group and control siRNA-transfected group in Jurkat cells ( Fig. 3 B and C).
To further explore its molecular mechanisms, we focused our study on several known G0/G1 phase cell cycle regulatory factors. Consistent with cell cycle arrest, cyclin D1 and CDK2 protein expression were decreased while p21 protein expression was increased ( Fig. 4 A and B). In accordance with the above cell cycle kinetics experiment, there was no significant change of the expressions of cyclin D1, CDK2 and p21 proteins in Jurkat cells ( Fig. 4 A and B).
3.4. Down-regulation of Notch-1 expression causes apoptosis in SupT1 cells
We next investigated whether the proliferation inhibition were related to the induction of apoptosis. Results showed that the early and late apoptosis rate was 9.01% and 12.41% in Notch-1 siRNA-transfected group, compared with 4.13% and 4.84% in control siRNA-transfected group, respectively ( Fig. 5 A). There was no apparent change in early and late apoptosis of Jurkat cells ( Fig. 5 B).
These data suggest that the growth inhibition induced by Notch-1 siRNA is partially due to the increased apoptosis in SupT1 cells.
3.5. Down-regulation of Notch-1 expression increases adriamycin-induced apoptosis in SupT1 cells
To further study whether down-regulation of Notch-1 expression altered the response of leukemic cells to chemotherapy, we have used siRNA to specifically knock down the expression of Notch-1 in SupT1 and Jurkat cells, and then examined cell viability and drug-induced apoptosis. Results showed that the IC50 value of adriamycin in SupT1 cells transfected with control siRNA was 0.21 μg/ml and SupT1 cells transfected with Notch-1 siRNA was more sensitive, with an IC50 value of 0.05 μg/ml ( Fig. 6 A). No marked difference was observed between Notch-1 siRNA-transfected group and control siRNA-transfected group in Jurkat cells ( Fig. 6 B).
In addition, we detected the rate of apoptosis induced by adriamycin after down-regulation of Notch-1. Results showed that the combination of Notch-1 siRNA and adriamycin group increased the apoptosis rate 2.4-fold in comparison with the combination of control siRNA and adriamycin group in SupT1 cells ( Fig. 7 A), while there was no significant difference in Jurkat cells between the combination of Notch-1 siRNA and adriamycin group and the combination of control siRNA and adriamycin group ( Fig. 7 B).
Furthermore, analysis of cell cycle following down-regulation of Notch-1 expression showed a marked G0/G1 phase arrest in SupT1 cells (P < 0.01, CS group vs. NS group) ( Table 2 ). Meanwhile, results showed that the combination of Notch-1 siRNA and adriamycin group increased G0/G1 phase cells in SupT1 cells (P < 0.01, CS + drug group vs. NS + drug group) ( Table 2 ). Nevertheless, there was no significant difference between the combination of Notch-1 siRNA and adriamycin group and the combination of control siRNA and adriamycin group in Jurkat cells ( Table 2 ).
|SupT1 (%)||Jurkat (%)|
|CS||21.17 ± 5.33||51.50 ± 7.22||27.33 ± 2.47||48.86 ± 4.37||44.88 ± 5.24||6.27 ± 1.34|
|NS||45.85 ± 6.65**||36.33 ± 5.24||17.27 ± 1.45||51.88 ± 6.56||44.30 ± 3.57||3.83 ± 2.94|
|Drug||14.07 ± 4.51||37.63 ± 2.34||48.30 ± 6.76||57.03 ± 5.34||32.33 ± 4.28||10.64 ± 2.36|
|CS + drug||16.59 ± 3.76||50.17 ± 3.57||33.24 ± 6.65||62.18 ± 6.39||37.76 ± 5.38||0.06 ± 2.64|
|NS + drug||36.53 ± 4.69#||45.49 ± 5.23||17.98 ± 9.37||59.39 ± 5.68||39.42 ± 5.61||1.18 ± 1.57|
Note: Flow cytometry was done as described in Section 2 . The indicated T-ALL cells transfected with control siRNA or Notch-1 siRNA were treated with or without adriamycin (0.1 μg/ml) for 48 h and cell cycle was assessed by flow cytometry using PI staining. CS, control siRNA; NS, Notch-1 siRNA; Drug represents adriamycin. #Statistically significant differences from control siRNA-transfected cells exposed to drugs (CS + drug) (P < 0.01). **Statistically significant differences from control siRNA-transfected cells (CS) (P < 0.01). The mean values (±S.E.) represent the percentage of cells in the indicated phase of the cell cycle from three independent experiments.
Adriamycin as a cell cycle-nonspecific drug also produced significant toxicity to G0/G1 phase cells induced by down-regulation of Notch-1 expression. Overall, Notch-1 siRNA-transfected SupT1 cells were significantly more sensitive to adriamycin-induced apoptosis.
3.6. Down-regulation of Notch-1 expression affects the level of p-Akt protein
Next p-Akt protein expression was examined by Western blot. Results showed that the expression of p-Akt protein of Notch-1 siRNA-transfected group was decreased in SupT1 cells ( Fig. 7 C). However, there were no such changes in Jurkat cells ( Fig. 7 C). Overall, down-regulation of Notch-1 expression in SupT1 cells increased apoptosis possible through the regulation of Akt signaling.
According to a recent study, more than 50% of all T-ALL patients carry Notch-1 gain-of-function mutations  . However, GSIs seem to be active in only a small fraction of human and mouse T-ALL cell lines with constitutive Notch-1 activity. Furthermore, gamma-secretase might affect other proteins. Therefore, in our study we make use of a more precise strategy, siRNA technology, to evaluate the role of Notch-1 in cell proliferation and apoptosis and explore its effective downstream target genes in two GSIs-resistant T-ALL cell lines. Our results indicate that down-regulation of Notch-1 elicited a dramatic effect on growth inhibition, apoptosis induction and drug sensitivity in SupT1 cells.
Cell proliferation is tightly regulated by expression and activation of cell cycle-dependent cyclins, CDKs, and CDK inhibitors. The D-type cyclins together with their kinase partners play a key role in regulating G1 progression  . Cyclin E interacts with CDK2 in late G1, resulting in cell cycle progression through G1 phase. The CDK inhibitors p21Waf1/Cip1 and p27Kip1 can inactivate cyclin D/CDK4,6 and cyclin E/CDK2 complexes resulting in G1 cell cycle arrest. Dohda et al.  reported that Notch/SKP2/p27Kip1 axis regulated G1/S cell cycle transition in T-ALL cells. To further understand why down-regulation of Notch-1 induced cell cycle arrest, we examined the expression of some other important cell cycle regulatory proteins. As expected, cyclin D1 and CDK2 expressions were decreased whereas p21 expression was increased in SupT1 cells. These findings corroborate recent data  and  that Notch-1 regulates the expression levels of some cyclins and CDK inhibitors and consequently the G1/S cell cycle transition.
Our study also revealed an interaction between Notch signaling and PI3K/Akt system. It is well known that PI3K/Akt system plays a critical role in cell proliferation, cell cycle progression and apoptosis in many human cancers. PI3K/Akt pathway activation is required for G1/S progression and PI3K/Akt inhibition leads to G1 arrest in many cell types , , and . Hyper-activated Akt kinases have been also shown to promote cell proliferation and cell cycle progression, possibly through up-regulation and stabilization of cyclin D1  and . It is evident from our work that down-regulated Notch-1 expression inhibits the PI3K/Akt pathway in SupT1 cells. It maybe an important mechanism of affecting cell cycle regulatory proteins and inducing G0/G1 phase cell cycle arrest by down-regulation of Notch-1. Hyper-activation of Akt kinases has also been found to mediate tumor cell survival and resistance to apoptosis  and . Notch-1 disrupts activated Akt signaling function and thus appears to be critical for apoptosis to occur  . In addition to the reports that Notch-1 inhibited drug-induced or p53-mediated apoptosis through PI3K/Akt pathway  and  and its inhibition reversed chemo-resistance  , our study presented a direct evidence of interaction between these signaling pathways in SupT1 cells.
Both SupT1 and Jurkat cells are GSIs-resistant T-ALL cells , , and , however, the two cell lines exhibited markedly different characteristics after down-regulation of Notch-1 expression by siRNA in our study. The reason of GSIs-resistance for SupT1 is that SupT1 cells aberrantly express an intracellular constitutively active form of Notch-1. The use of Notch-1 siRNA easily overcomes this obstacle and allows the observation that Notch-1 is important for SupT1 maintenance. In contrast, Jurkat cells are insensitive to GSIs for another reason. The activating mutations in Notch-1 present in Jurkat cells should theoretically render Jurkat cells sensitive to GSIs. However, they lack PTEN and thus have constitutive PI3K/Akt pathway hyperactivation. According to Palomero et al.  , this results in a shift in ‘oncogene addiction’ from Notch-1 signaling to PI3K/Akt pathway signaling, ultimately leading to sensitivity to PI3K inhibitors and resistance to GSIs treatment. Therefore, Notch-1 down-regulation by siRNA is also ineffective as demonstrated in our study. It is noted that the basal level of p-Akt is very similar in SupT1 and Jurkat cells ( Fig. 7 C), indicating that, despite expressing PTEN  , SupT1 cells have levels of PI3K/Akt pathway activation similar to PTEN-deficient Jurkat cells. This observation is not consistent with the ‘oncogene addiction shift’ model proposed by Palomero et al.  and calls for further investigation on the biological and clinical significance of the crosstalk between Notch-1 and other signaling pathways.
Mutations in the Notch-1 signaling pathway have emerged as a frequent genetic component in T-ALL. However, the prognostic significance of Notch-1 mutations remains controversial. Breit et al. reported that mutations were associated with a favorable outcome  , but in the majority of studies, it was found to contribute to poor outcome  and . More recently, it was shown that Notch-1 mutations had no prognostic significance  . Maybe the different number and race of patients or the various regions of Notch-1 mutations result in the discrepancy. Therefore, further studies with larger cohorts of patients are warranted to evaluate the prognostic significance. In view of frequent Notch mutations and GSIs resistance, elucidation of the Notch downstream response is important as it could be a basis for future improved treatments.
In conclusion, our study indicates that Notch-1 plays an important role in T-ALL cell proliferation, cell cycle progression and apoptosis. Our results also imply that cell cycle regulatory proteins and Akt signaling may be attractive therapeutic targets in T-ALL.
Conflict of interest
There is no conflict of interests.
This work was supported by Grant 30471941 from National Nature Science Foundation of PR China and Grant 20060422051 from Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) and Grant 03BS025 and 2005GG4202018 from Bureau of Science and Technology of Shandong Province, PR China.
Contributions. D. Guo contributed to the concept and design, data analysis and interpretation, statistical expertise, drafting of the article, collected and assembled the data and gave final approval. J. Ye contributed to the concept and design, provided critical revisions, provided technical support and gave final approval. J. Dai contributed to the study design, collected and assembled the data, data analysis and interpretation, and gave final approval. L. Li contributed to technical support, helped to analyze the data, and gave final approval. F. Chen contributed to collect and assemble the data, statistical expertise and gave final approval. D. Ma contributed to the data analysis and interpretation, drafting of the article, provided critical revisions to the revision and gave final approval. C. Ji contributed to the study design, gave critical input to the revision, obtained funding, gave final approval and provided administrative support.
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